Cold Atmospheric Plasma Stimulates Clathrin-Dependent Endocytosis to Repair Oxidised Membrane and Enhance Uptake of Nanomaterial in Glioblastoma Multiforme Cells

Zhonglei He,Brijesh Tiwari,Laurence Scally,Patrick J Cullen,Furong Tian,James F Curtin,Hugh J Byrne,Sebnem Gunes,Sing Wei Ng,Marcus Maher,Eline Manaloto,Paula Bourke,Kangze Liu

doi:10.1038/s41598-020-63732-y

Abstract

Cold atmospheric plasma (CAP) enhances uptake and accumulation of nanoparticles and promotes synergistic cytotoxicity against cancer cells. However, the mechanisms are not well understood. In this study, we investigate the enhanced uptake of theranostic nanomaterials by CAP. Numerical modelling of the uptake of gold nanoparticle into U373MG Glioblastoma multiforme (GBM) cells predicts that CAP may introduce a new uptake route. We demonstrate that cell membrane repair pathways play the main role in this stimulated new uptake route, following non-toxic doses of dielectric barrier discharge CAP. CAP treatment induces cellular membrane damage, mainly via lipid peroxidation as a result of reactive oxygen species (ROS) generation. Membranes rich in peroxidised lipids are then trafficked into cells via membrane repairing endocytosis. We confirm that the enhanced uptake of nanomaterials is clathrin-dependent using chemical inhibitors and silencing of gene expression. Therefore, CAP-stimulated membrane repair increases endocytosis and accelerates the uptake of gold nanoparticles into U373MG cells after CAP treatment. We demonstrate the utility of CAP to model membrane oxidative damage in cells and characterise a previously unreported mechanism of membrane repair to trigger nanomaterial uptake. This knowledge will underpin the development of new delivery strategies for theranostic nanoparticles into cancer cells.

Highlights

AuNPs have emerged as a promising reagent, combined with Cold atmospheric plasma (CAP), for anti-cancer therapy[4,12,13]
We demonstrate, for the first time, that synergistic effects of nonfunctionalised AuNPs combined with CAP is via clathrin-mediated endocytosis pathway and is triggered as part of the CAP-induced membrane repair process in Glioblastoma multiforme (GBM) cells
U373MG cells was monitored using atomic absorption spectroscopy and the dose response curve of AuNPs with or without CAP treatment has been presented in previous study[26]

Summary

Introduction

AuNPs have emerged as a promising reagent, combined with CAP, for anti-cancer therapy[4,12,13]. Several ROS and RNS generated by CAP can induce cell injures via lipid peroxidation[22], for instance, hydroxyl radicals (OH) react with various cellular components, including membrane lipid[23] while superoxide (O2−) can form peroxynitrite (ONOO−), which is able to initiate lipid peroxidation, after reacting with nitric oxide (NO)[23]. We demonstrated CAP in combination with AuNPs shows promising synergistic cytotoxicity to U373MG Glioblastoma multiforme (GBM) cells and ATP-dependent uptake mechanism[26]. Kim et al demonstrated that epidermal growth factor-conjugated AuNPs combined with CAP induced DNA damage and selective apoptosis of A549 human cancer cells after uptake via receptor-mediated endocytosis[25]. We demonstrate, for the first time, that synergistic effects of nonfunctionalised AuNPs combined with CAP is via clathrin-mediated endocytosis pathway and is triggered as part of the CAP-induced membrane repair process in GBM cells. Our use of CAP as an alternative novel tool to trigger and understand membrane oxidative damage and repair mechanisms is outlined

Methods

Results

Conclusion